In first step of insulin action, insulin binds to its receptor on the surface of the target cell. The insulin receptor is a transmembrane protein which possesses a tyrosine-specific proteins kinase. When insulin binds to the extracellular domain of the receptor, this activates the receptor's tyrosine kinase. A growing body of evidence suggests that the activation of the tyrosine kinase is a necessary step in initiating the biological actions of insulin. Accordingly, we have embarked upon a search for intracellular proteins which are substrates for phosphorylation by the receptor-associated tyrosine kinase. We have identified one such substrate in rat liver plasma membranes: a glycoprotein with an apparent molecular weight of 120,000 daltons (pp120). pp120 is present in liver from several species, but has not been identified in other tissues. We have demonstrated that pp120 is identical to HA4, a glycoprotein localized to the bile canalicular domain of the hepatocyte plasma membrane. Monoclonal antibodies to HA4 have been used to immunoaffinity purity HA4/pp120. We are obtaining partial amino acid sequences of purified HA4/pp120. This amino acid sequence data will be used to clone cDNA encoding HA4/pp120.